Human amniotic epithelial cells:isolation, identification and multi-directional differentiation / 中国组织工程研究

ABSTRACT

BACKGROUND: Human placenta is a stable source for human amniotic epithelial cells, which is becoming a cellsource in the regenerative medicine that attracts widespread attentions.OBJECTIVE: To establish the method of isolation, culture, and adipogenic, chondric and osteogenic differentiationof human amniotic epithelial cells. METHODS: Trypsin-EDTA digestion was used to isolate human amniotic epithelial cells from human amnion tissue,which were then cultured and identified in vitro. The growth curve of the cells was observed in 12 days. Passage 1human amniotic epithelial cells were induced to differentiate into adipocytes, chondrocytes and osteoblasts, andconventional cultured cells were used as controls. After 16 days induction, oil red O, Masson and alkaline phosphatesstaining methods were carried out, and adipogenic transcription factor, type Ⅱ collagen, osteopontin, alkalinephosphatase mRNA expressions were detected using real-time fluorescene quantitative PCR.RESULTS AND CONCLUSION: Human amniotic epithelial cells were successfully obtained from human amnion tissue.Immunofluorescence data showed the expression of epithelial cell surface marker CK19. Passage 1 cells had a strongability to divide and proliferate. Compared with passage 1 ones, passage 2 cells showed a slight decrease in proliferationability, and the proliferation ability of passage 3 cells was the worst. Red lipid droplets, brilliant blue cartilage matrix andreddish brown calcium nodes were detected by oil red O, Masson and alkaline phosphates staining after adipogenic,chondrogenic and osteogenic differentiation, respectively. With the time prolonged, the expressions of adipogenictranscription factor, type Ⅱ collagen, osteopontin and alkaline phosphatase mRNA were increased. These resultsdemonstrated that human amniotic epithelial cells could be isolated from human amniotic membrane by enzymedigestion method, and these amniotic epithelial cells could be induced to differentiate into differentiate into adipocytes,chondrocytes and osteoblasts.